Project Summary Streptococcus pneumoniae normally colonizes the nasopharynx of healthy individuals yet is a major respiratory pathogen among the elderly that causes pneumonia, septicemia, and meningitis, despite the availability of two vaccines against S. pneumoniae. One reason the elderly are more susceptible to S. pneumoniae infection is because innate and adaptive immunity, particularly that of T cell-mediated function, declines with age; when faced with S. pneumoniae infection, the aged host cannot mount an appropriate response against the bacteria and fails to control the infection, resulting in large amounts of pulmonary inflammation and invasive disease. Importantly, oral supplementation with vitamin E can reverse the age-driven susceptibility to S. pneumoniae infection in aged mice, and reverses age-related defects in T cell signaling and proliferation in vivo. The T cell- dependent pneumococcal conjugate vaccine (PCV13) appears to provide higher protection to the elderly than the T cell-independent pneumococcal polysaccharide vaccine (PPSV23). Nasopharyngeal (NP) colonization in healthy adults and mice also acts as a T cell-dependent immunizing event, implicating the critical role of T cells during development of an adaptive immune response against S. pneumoniae. To further understand age- related defects of innate and adaptive immune responses against this pathogen, S. pneumoniae factors required for early survival in young or vitamin E-supplemented aged mice but not untreated mice will be identified, providing insight into the innate immune selective pressures and defects of an aged host (Aim 1), and it will be determined if vitamin E supplementation reverses defects of T cell function and differentiation in an aged host (Aim 2). To identify classes of S. pneumoniae genes that are required for survival in immunocompetent (young mice or vitamin E-supplemented aged) mice but not immunocompromised, aged mice, Tn-seq, a high-throughput genetic selection method designed to identify these factors, will be utilized. To determine if vitamin E restores protection to aged mice through a T cell-dependent mechanism, young and aged mice fed a control or supplemented vitamin E diet will be NP colonized with S. pneumoniae or vaccinated with PCV13 or PPSV23. Development of immune responses in each group will be analyzed by measuring anti- pneumococcal antibody titers and T cell proliferation and differentiation. Ultimately, disease outcome after S. pneumoniae challenge will provide insight on the ability of vitamin E to improve protective T cell-mediated adaptive immunity in aged mice. This proposal aims to further investigate the age-associated defects of the innate and adaptive immune responses to S. pneumoniae and may impact S. pneumoniae vaccine development and dietary guidelines for older adults to prevent pneumococcal disease.